The following is a discussion of the relevant art, none of which is admitted to be prior art to the appended claims.
A variety of methods have been developed to efficiently amplify DNA using purified proteins. These generally can be classified as ones that use thermocycling of the reaction mixtures and ones in which the reactions are isothermal. Methods that use thermocycling of the reaction mixture are based on the polymerase chain reaction, or PCR (Saiki et al., Science 230:1350, 1985). In PCR, two primers are added to the target DNA, such that the two primers are complementary to opposite strands of the target sequence with their 3′ ends oriented towards one another. Multiple cycles of denaturation of target DNA, annealing of the two primers, and then extension of the primers with a DNA polymerase, results in an exponential synthesis of the target DNA sequence located between the two primers. This procedure requires the use of a DNA polymerase that is thermostable in order to survive the high temperatures required to denature the product DNA each cycle. The most common polymerase used for PCR is from Thermus aquaticus, or Taq DNA polymerase (Saiki et al., Science 239:487, 1988). Modification of the reaction conditions and the enzyme mixture used can allow the amplification of DNA up to 50 kbp in length (Barnes, Proc. Natl. Acad. Sci. USA 91:2216, 1994 and Cheng et al., Proc. Natl. Acad. Sci. USA 91:5695, 1994).
Isothermal procedures for the amplification of nucleic acid include (1) Qβ replicase, (2) self-sustained sequence replication (3SR), (3) strand displacement amplification (SDA), (4) terminal protein-primed DNA amplification using Ø29 DNA polymerase, and (5) rolling circle amplification (RCA).
The RNA-dependent RNA polymerase Qβ polymerase has been used for gene detection in a strategy that uses two probes that hybridize to adjacent positions on a target sequence that are then ligated to form an amplifiable reporter RNA (Tyagi et al., Proc. Natl. Acad. Sci. USA 93:5395, 1996).
Self-sustained sequence replication (3SR) is a method of amplifying RNA that depends on the action of three enzymes; reverse transcriptase, DNA-dependent RNA polymerase and ribonuclease H (Guatelli et al., Proc. Natl. Acad. Sci. USA 87:1874, 1990).
Strand displacement amplification (SDA) is a DNA amplification system that uses a restriction enzyme to introduce specific nicks in a target to be amplified and a DNA polymerase that is capable of strand displacement synthesis (Walker et al., Proc. Natl. Acad. Sci. USA 89:392, 1992).
Terminal protein-primed DNA amplification exponentially amplifies linear Ø29 DNA using the Ø29 proteins DNA polymerase, terminal protein, double-stranded DNA binding protein, and single-stranded DNA binding protein (Blanco et al., Proc. Natl. Acad. Sci. USA 91:12198, 1994). To date this method has only been successful using linear Ø29 DNA as template.
Rolling circle amplification (RCA) can either be linear or exponential amplification of circular oligonucleotide probes that use two primers that anneal to each of the two strands, and a DNA polymerase that strand displaces (Lizardi et al., Nature Genetics 19:225, 1998 and Zhange et al., Gene 211:277, 1998).
In order to be used as a generalized amplification system, all of these methods require the addition of specific oligonucleotide primers that are extended by a polymerase; the primers serve to fix the limits of the segment of nucleic acid to be amplified.